The mechanism of transport of the multitargeted antifolate (MTA) and its cross-resistance pattern in cells with markedly impaired transport of methotrexate

MTA (LY231514) is an antifolate that targets multiple folate-dependent enzy mes. In this report, MTA transport was characterized in wild-type L1210 cel ls and variants with impaired membrane transport or polyglutamation, MTA in flux via the reduced folate carrier was somewhat faster (similar to 30%) th an that for methotrexate (MTX), Unlike MTX, MTA was rapidly polyglutamated in L1210 cells; hence, a folylpoly-gamma-glutamate synthetase-deficient L12 10 variant was used to assess net transport and efflux properties. The MTA transmembrane gradient for exchangeable drug was 2.5 times greater than the MTX gradient, attributable primarily to an efflux rate constant 40% that o f MTX, No MTA was bound to dihydrofolate reductase, When grown with folic a cid, MTX-resistant L1210 variants with mutations in the reduced folate carr ier demonstrated cross-resistance to MTA, markedly reduced MTA accumulation , and only a slightly decreased intracellular folate cofactor pool as compa red to L1210 cells. However, when 5-formyltetrahydrofolate was the growth s ubstrate, these MTX-resistant cells were less resistant or negligibly resis tant to MTA, accumulated more MTA, and had a lower folate pool as compared to L1210 cells. MTA activity and the intracellular folate pool in L1210 cel ls were inversely related. These data indicate that MTA polyglutamation in L1210 cells is favored by both the generation of high intracellular drug le vels and high MTA affinity for FPGS relative to MTX, Cells resistant to MTX because of impaired transport may retain appreciable sensitivity to MTA be cause of a concurrent reduction in tetrahydrofolate cofactor transport resu lting in cellular folate depletion, which diminishes endogenous folate supp ression of MTA polyglutamation.